This laboratory previously described a single‐laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria‐infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra‐ and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria‐infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra‐ and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei. Environ. Mol. Mutagen. 38:59–68, 2001 © 2001 Wiley‐Liss, Inc.

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

In collecting inhalation toxicity data for the evaluation of the health hazard from occupational exposure to the aerosols of a drug or a chemical, the determination of the inhaled dose in relation to the animal response is most desirable. Intratracheal administration is most likely to deliver an exact dose of a compound to the lungs of an experimental animal. In a series of tests, microliter (mu l) quantities of a solution or a suspension of a test material were nebulized into the trachea of an anesthetized rat using an intratracheal fast instillation (ITFI) method. The dose-response in terms of the minimal effective dose (MED) and the median lethal dose (LD50) were determined. The ITFI dose-response for four drugs, five chemicals or chemical intermediates and four pesticides were compared with those obtained via inhalation (IH) and ingestion (p.o.), In addition, the dose-responses of the four pesticides were compared with two additional parameters, intranasal instillation (IN) and intravenous injection (i.v.), The MED end-points for studies via the respiratory administration route were no pharmacotoxic signs other than transient respiratory rales and/or dyspnea and no gross lesions, whereas those for the intranasal, oral and the intravenous administration routes were transient and slight body weight loss and no pharmacotoxic signs and/or gross lesions. The MED ratios between ITFI, In and p.o. were 1 : 9.3 +/- 6.5 : 201.4 +/- 133.3, respectively, for the drugs, chemicals and chemical intermediates. The MED ratios for ITFI, IH, IN, i.v. and p.o. for the four pesticides were 1 : 2.2 +/- 1.4 : 2.1 +/- 1.3 : 1.1 +/- 0.7 : 1.4 +/- 0.9. The MED ratios for the two categories of test materials were fairly consistent between different routes of administration. Thus, the ITFI dose can be used for extrapolating the IH dose. The simplicity of the ITFI procedure and its requirement of only microliters of a compound to generate a meaningful and reliable dose-response suggests that ITFI may be an alternative method for acute inhalation toxicity evaluation of materials that may present inhalation hazards from liquid or solid aerosols, Copyright (C) 1999 John Wiley & Sons, Ltd.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073–9078] to the previously established Big Blue® assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue® mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 × 10−5 for cII/cI vs. 15.1 × 10−5 for lacI; spleen = 64.8 × 10−5 for cII/cI vs. 36.1 × 10−5 for lacI; lung = 17.1 × 10−5 for cII/cI vs. 15.8 × 10−5 for lacI). Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs. 2.7 for cII/cI; spleen = 13.1-fold for lacI vs. 8.4 for cII/cI; and lung = 5.6-fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated. Environ. Mol. Mutagen. 33:249–256, 1999 © 1999 Wiley-Liss, Inc.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.

The effectiveness of three techniques to deliver a diazo dye suspension into the lungs of rats was compared. The intratracheal nebulization (ITN) technique delivered 10 μl of the suspension per 5-ml puff of air in 10 puffs as an aerosol. The intratracheal fast instillation (ITFI) technique delivered 100 μl of the suspension in a single 2-ml puff of air as droplets. The nose-only inhalation (NI) technique aerosolized the suspension at an analytical concentration that provided a calculated dose equivalent to 100 μl of the suspension in a 2-h inhalation period. Immediately after dosing, all the rats were killed by exsanguination. The trachea was tied and the lung was inflated in situ with air. After fixation, 5-μm thick slices were prepared from each lobe of the lung at a plane perpendicular to the axis of the lobar bronchus at levels proximal, medial and distal to the hilus. The numbers of bronchi, bronchioli and alveolar ducts within four ranges of diameters and the proportion of each selected area of lung tissue with and without dye particles were quantified using electronic imaging analyzers. The results indicated that ITN and ITFI dispersed the particles evenly throughout most of the airways and in patches in the alveoli. The NI technique dispersed the particles homogeneously throughout the airways and the alveoli in the lungs. The mean number-percentage and the mean area-percentage data revealed that the doses delivered by ITN and NI were approximately 60% and 10%, respectively, of the ITFI dose. Thus, the ITFI technique appeared to be most suitable for pulmonary absorption and disposition studies where dosage precision is of primary concern. The ITN technique would need further improvement to meet the requirements for dose precision and particle distribution. For both ITFI and ITN, particle size was apparently not a critical determinant for deposition. The NI technique is suitable for inhalation toxicity studies where the pattern and uniformity of particle deposition is the primary concern. © 1998 John Wiley & Sons, Ltd.

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.

We have monitored mutant frequency at the HPRT locus in peripheral blood lymphocytes of cynomolgus monkeys using a clonal assay in which mutants are selected by resistance to 6‐thioguanine. Among untreated animals, the mean spontaneous mutant frequency was 2.9 ± 2.9 × 10‐6 (standard deviation, based on 131 determinations in 33 animals), in good agreement with HPRT mutant frequencies in other species. In four animals treated with a single intraperitoneal dose of 77 mg/kg ethylnitrosourea, mutant frequency increased with time, peaking 70 to 100 days after treatment. Mutant frequency in two of the four animals was monitored at intervals for 6 years, and a second identical treatment was given about 830 days after the first. Mutant frequency again peaked in these two animals 70 days after the second dose and decreased following peak values, declining to a plateau that was higher than the predose mutant frequency in both animals. This pattern was repeated following the second ethylnitrosourea treatment. Fractionating the dose of ethylnitrosourea into five equal daily injections had no effect on mutant frequency in two animals when compared to a single dose. Environ. Mol. Mutagen. 29:117‐123, 1997. © 1997 Wiley‐Liss, Inc.

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1–40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Estimation of population exposure and biological impact of potential hazards are central reasons for performing biomonitoring. The sensitivity of the biomonitoring methods and the linkage of the measured phenomenon to human disease are also important, but often overlooked, considerations. We are conducting experiments to evaluate the sensitivity of hprt mutation measurement in the nonhuman primate, the cynomolgus monkey. Our findings demonstrate in the monkey that hypoxanthine guanine phosphoribosyltransferase (hprt) mutations produced in vivo can be detected using technique originally worked out using human cells; cynomolgus monkeys were chosen to avoid many of the complications encountered in studying humans. Sequencing of mutants from the monkey using reverse transcriptase polymerase chain reaction methods has led us to conclude that there is similarity of the spectra observed between the spontaneous mutations detected in the two species. However, more recent data suggest that due to low sensitivity, the method is probably not appropriate for routine biomonitoring of randomly selected populations. For example, the inability of the hprt mutation assay to detect some very potent mutagens in the monkey and the effects of the time-dependent pattern of mutant occurrence serve to urge caution in interpretation of elevation or lack of elevation in mutant frequency. Mechanisms for splitting and archiving samples of human tissues/blood from populations at risk may prove valuable as methods improve.

Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5). Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

The in vivo genotoxic effects of the antitumor antibiotic, (+)-CC-1065, and its unnatural enantiomer, (-)-CC-1065, were investigated in two mouse models. These two compounds alkylate AT-rich regions of double stranded DNA with distinct sequence selectivities. (+)-CC-1065 dose-dependently increased the chromosomal aberration frequency in bone marrow cells of CD-1 mice from 1.2 +/- 0.8% in vehicle control animals to 5.0 +/- 1.2%, 11.4 +/- 3.9%, and 20.6 +/- 2.3% 24 hours following single intravenous doses of 2, 4, and 8 micrograms/kg, respectively. (-)-CC-1065 was significantly less potent with a maximal response at 8 micrograms/kg approximately one-third of that observed for (+)-CC-1065. (+)-CC-1065 induced a significant (P < or = 0.05), three-fold increase in the number of lung tumors/mouse in strain A/J mice from 0.27 +/- 0.15 for vehicle control animals to 0.83 +/- 0.15 24 weeks following a single intravenous dose of 8 micrograms/kg. This effect was paralleled by corresponding threefold increases in the percentage of mice with tumors and the percentage of mice with multiple tumors, compared to vehicle controls. (-)-CC-1065 at 8 micrograms/kg induced 0.67 +/- 0.15 tumors/mouse and resulted in slightly smaller increases in the tumor incidence and multiple tumor incidence, compared to (+)-CC-1065. The above results demonstrate that single intravenous doses of (+)- CC-1065 and (-)-CC-1065 which cause chromosomal damage in CD-1 mice also induce an increased incidence of lung tumors in A/J mice.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T‐lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wild‐caught cynomolgus monkey peripheral T‐lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6‐thioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780‐base‐pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monkeys. Of these 40 clones, 23 (57%) had base‐pair substitutions, 11 (28%) had small (<20 bp) deletions and/or insertions, and 6 (15%) had large (>20 bp) deletions and/or insertions. Of the 23 base substitutions, 13 were transitions (11 G:C → A:T, 1 A:T → G:C, and 1 tandem TT → CC) and 10 were transversions (3 G:C → T:A, 3 G:C → C:G, 2 A:T → T:A, 2 A:T → C:G). Bases 209 and 617 were apparent substitution hotspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic origin. One of these lost 272 bp from exons 2–3 and contained a 93‐bp insertion from the middle of intron 3. Two clones with small deletions and 5 clones with large deletions or insertions (7/40 or 17.5%) could be splice mutants. © 1995 Wiley‐Liss, Inc.

Ceftiofur, a new generation of cephalosporin antibiotic, used to combat bacterial respiratory disease in growing cattle and swine, has been tested in a battery of genetic toxicology assays (Aaron et al., 1995a) and been shown to produce chromosome aberrations in CHO cells following treatment for 44 h. No evidence of aberration induction was seen at shorter, i.e., 20 h, treatment time nor was any suggestion of clastogenicity seen in the presence of S9 metabolic activation. The experiments reported here were undertaken to determine significance of this observation and elucidate the reason for clastogenesis in the earlier experiments. Briefly, ceftiofur was found to not affect the pH or osmolality of the treatment solutions nor were enzymes generally associated with cell death released during the treatment period. The aberrations were found to be reversible, and thus, doubt was cast concerning the potential for direct DNA damage as a causative factor. The most profound effect of ceftiofur treatment at this level was the dramatic effect on cell cycle kinetics and therefore the clastogenic effects observed following exposure to cettiofur in vitro appear to be due to prolongation of the cell cycle.

Ceftiofur has been previously shown (Aaron et al., 1995a Mutation Res., this issue) to produce chromosome aberrations following extended treatment of Chinese hamster ovary cells in culture at high doses in the absence of S9 activation. The experiments described here address the relevance of this finding in vivo. Ceftiofur failed to induce aberrations in the bone marrow of CD-1 mice following intraperitoneal injection. Furthermore, ceftiofur did not induce micronucleated polychromatic erythrocytes in mouse bone marrow. In the experiments reported here no evidence of the induction of unscheduled DNA synthesis in the liver of rats treated in vivo was seen following oral exposure to high doses of the drug. However, the compound was foud to reach the bone marrow compartment in these experiments. Thus, the negative finding suggests that the in vitro finding of chromosome aberration induction by ceftiofur is not a cause of concern. This assertion is supported by mechanistic studies (Aaron et al., 1995b, Mutation Res., this issue) which show the cause of the in vitro findings to be most likely due cell cycle delay and by the observation that ceftiofur is rapidly metabolized in mammalian systems in vivo but not in the artificial conditions that occur in the in vitro experiments.

Preclinical safety evaluation of new drugs is routine prior to the use in humans or animals and genetic toxicology assays are an accepted part of the evaluation along with other more traditional measures of toxicity. A widely used battery of genetic toxicology assays includes an Ames Salmonella microsome assay, a mammalian cell mutation assay, a rat bone marrow micronucleus test and an in vitro assay for induction of chromosomal aberrations. Ceftiofur (U-6427E, NAXCEL®, EXCENEL®), a new generation cephalosporin antibiotic, was subjected to this battery of assays. The result of the first three (Ames test, V79/HPRT mammalian cell mutation assay and the micronucleus test) were negative, the in vitro assay for induction of chromosome aberrations in CHO cells gave positive results. The nature of the observed aberrations was primarily chromatid and isochromatid breaks, gaps and fragments with little evidence of chromosomal rearrangements in the absence of S9 metabolic activation. The aberration increase was only seen following an extended (44 h) exposure to drug; no evidence of clastogenic activity was seen in the presence of S9 metabolic activation or at shorter treatment times. The severe treatment conditions required to produce the clastogenic effects also produced very high levels of mitotic inhibition and thus the observation of chromosomal aberrations is unlikely to be biologically meaningful.

Increases in peripheral blood T‐lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T‐cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR B and y genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 × 10 −6 , compared to the normal mean MF of 3.03 × 10 −6 . The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T‐cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR y along with an apparent germline TCR B configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U‐73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR y and B genes. Possible explanations for these findings are discussed. © 1995 Wiley‐Liss, Inc.

Monocrotaline is a very potent toxin, producing significant effects of pneumotoxicity, hepatotoxicity, and teratogenicity, as well as carcinogenicity. In addition, the compound has been clearly shown to be mutagenic after metabolic activation. The goal of the experiments reported here was to confirm the reported clastogenesis induced by this agent in vivo and to evaluate the impact of modulation of metabolic activity by phenobarbital, a potent P-450 inducer (both Phase I and Phase II enzymes). The method used in addressing this problem relied on a new technique For monitoring clastogenesis in vivo, i.e., the acridine orange micronucleus assay method originally exploited by Hoyashi et al. [1990].The result of our experiments confirmed monocrotaline to be an effective clastogen in vivo, using the acridine orange method of assessment. The peak in induction of micronuclei occurred on the second day following intraperitoneal administration of the drug. Administration of phenobarbital prior to monocrotaline did appear to modulate the micronucleus induction. At 30 mg/kg bw monocrotaline, the pretreatment with phenobarbital appears to increase the intensity of monocrotaline clastogenesis, while the effect at higher doses (60 and 125 mg/kg bw) is a reduction in potency, presumably reflecting increased importance of Phase II metabolism for monocrotaline at these doses. Thus the Study reported here confirms the potent in vivo clastogenesis of monocrotoline, and provides evidence for a dose-related shift in mechanism for the phenomenon. (C) 1995 Wiley-Liss, Inc.

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